Use of imipramine blue and analogs thereof in treating cancers

ABSTRACT

Compounds, pharmaceutical compositions including the compounds, and methods of preparation and use thereof are disclosed. The compounds are triphenyl methane analogs. The compounds and compositions can be used to treat and/or prevent a wide variety of cancers, including drug resistant cancers, inflammatory, degenerative and vascular diseases, and parasitic infections. The compounds are triphenyl methane analogs of imipramine blue and analogs thereof, as defined herein. The compounds are believed to function by inhibiting tNOX expression, the effects of ROS, and/or the production of HIF2. Thus, the compounds are novel therapeutic agents for a variety of cancers and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.12/345,652, filed Dec. 29, 2008, which itself claims the benefit of U.S.Provisional Patent Application No. 61/009,745 filed Dec. 31, 2007, andU.S. Provisional Patent Application No. 61/090,027 filed Aug. 19, 2008.The disclosures of all of the foregoing applications are herebyincorporated herein in their respective entireties, for all purposes.

GOVERNMENT RIGHTS IN INVENTION

Some aspects of the invention disclosed in this application weresupported by the United States government, National Institute of HealthGrant No. AR02030. Accordingly, the U.S. Government has certain rightsin the invention hereof.

FIELD OF THE INVENTION

The present invention relates to novel methods and compositions for thetreatment of primary and metastatic cancers and other proliferativedisorders. These methods and compositions use a specific triaryl aminecompound, herein named imipramine blue, and analogs thereof. Thesecompounds, and pharmaceutical compositions including the compounds, areparticularly useful for treating primary and metastatic cancers inhumans. The invention also encompasses the varying modes ofadministration of the therapeutic compounds or compositions.

This invention was made with government support under grant numberAR02030 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Cancer is a multistep process, beginning with minorpreneoplastic changes, which may under certain conditions progress toneoplasia. Malignant endothelial tumors arise in the setting ofautocrine loops involving vascular endothelial growth factor (VEGF) andits major mitogenic receptor vascular endothelial growth factor receptor2.

Reactive oxygen species (ROS) are believed to be mediators of growth andangiogenesis in cancer. Increased ROS often correlates with cell growth,e.g., Ras-transformed cells and cells treated with growth factors. Whilenon-transformed cells respond to growth factors/cytokines with theregulated production of ROS, tumor cells in culture frequentlyoverproduce H₂O₂.

NAD(P)H oxidase (Nox) is a cell surface protein with hydroquinone (NADH)oxidase and protein disulfide-thiol interchange activities. In general,most forms of the enzyme can utilize either NADH or NADPH equallyefficiently. There are many forms of Nox, including Nox 1-5, Dualoxidase 1 and 2 (Duox 1 and 2), as well as p22(phox), p47(phox) and thesmall G-protein Rac1.

Nox are believed to account for increased levels of ROS in certaincancers. Reactive oxygen-generating Nox enzymes are implicated in theangiogenic switch, and Nox inhibitors have an effect on ang-2 productionin vitro and on bEnd.3 tumor growth in vivo. ang-2 production can beinhibited pharmacologically using Nox enzyme inhibitors, which nearlyabolishes bEnd.3 hemangioma growth in vivo. Signal-transduction blockadetargeting ang-2 production may therefore be useful for treating humanhemangiomas in vivo. Journal of Investigative Dermatology advance onlinepublication, 1 Jun. 2006; doi:10.1038/sj.jid.5700413.

With respect to specific Nox enzymes, it has been shown thattransfection of Nox1 into a prostate cancer cell line dramaticallyenhanced tumor growth (Arbiser et al.: PNAS 99:715-720, 2001), andprostate tumors show increased H₂O₂ levels. Further, prostate tumorswere recently found to show increased levels of Nox1 and hydrogenperoxide (Lim et al., Prostate. 2005 Feb. 1; 62 (2):200-7). Nox1-dependent superoxide production has also been shown to control colonadenocarcinoma cell migration (Sadok et al., Biochim Biophys. Acta. 1783(1):23-33 (January 2008). Sadok showed that Nox1 inhibition ordown-regulation led to a decrease of superoxide production and alpha 2beta 1 integrin membrane availability. Thus, there is a correlationbetween Nox protein levels and ROS in prostate cancer, and increasedNox1/H₂O₂ correlates with increased tumorigenicity.

Nox4 is believed to be implicated in inhibition of apoptosis in cancercells, such as pancreatic cancer cells (Vaquero et al., J Biol. Chem.2004 Aug. 13; 279 (33):34643-54). Vaquero suggested that growthfactor-induced ROS produced by NAD(P)H oxidase (probably Nox4) protectspancreatic cancer cells from apoptosis, and that transfection with aNox4 antisense oligonucleotide inhibited NAD(P)H oxidase activity andROS production in certain pancreatic cells (i.e., MIA PaCa-2 and PANC-1cells), and stimulated apoptosis in these cells.

Akt, a signaling molecule downstream of PI3K (phosphoinositol-3-kinase),is known to induce expression of the ROS-generating enzyme Nox4. Onestudy introduced Akt into a radial growth WM35 melanoma in order to testwhether Akt overexpression was sufficient to transform the cells fromradial growth to vertical growth. Overexpression of Akt led toupregulation of VEGF, increased production of superoxide ROS, and theswitch to a more pronounced glycolytic metabolism. Subcutaneousimplantation of WM35 cells overexpressing Akt led to rapidly growingtumors in vivo, while vector control cells did not form tumors. Arbiseret al., J. Invest. Dermatol. 2006, Jun. 1, 16741507. This data supportsthe premise that inhibition of Akt can inhibit downstream production ofNox 4, which then would inhibit superoxide generation, and thereforetreat melanoma.

Duox 1 and 2 are the major Nox species in airway endothelia, and arebelieved to be one of the main sources for reactive oxygen speciesproduction in the airway (Luxen et al., Cancer Res. 2008 Feb. 15; 68(4):1037-45). Accordingly, inhibition of these enzymes may be useful intreating human lung cancer.

Some authors have characterized Nox as falling into two categories. Oneis hormone-insensitive and drug-responsive (i.e., by quinine-siteinhibitors such as capsaicin or the antitumor sulfonylurea, LY181984),designated “tNox,” which is specific to cancer cells. The other is adrug-indifferent constituitive form associated with the plasma membraneof non-transformed cells, designated “CNox” (Kelker et al.,Biochemistry. 26; 40 (25):7351-4 (2001); Wang et al., Biochim BiophysActa. June 20; 1539 (3):192-204 (2001)

Cancer cells exhibit both drug-responsive and hormone and growthfactor-indifferent (tNox), and drug inhibited and hormone and growthfactor dependent (CNox) activities, whereas non-transformed cellsexhibit only the drug indifferent hormone- and drug-responsive CNox.Like the tNox of cancer cells, CNox is capable of oxidizing NADH, buthas an activity which is modulated by hormones and growth factors. Thus,some authors have theorized that inhibitors of tNox (which are believedto include one or more of the Nox enzymes listed above, such as Nox4)will be useful for treating cancer.

In addition to treating cancer, Nox inhibitors are also expected toprovide therapeutic effects for numerous other inflammatory,degenerative and vascular diseases in which reactive oxygen species havebeen implicated.

In non-ocular cutaneous tissues, NADPH oxidase from pollen has beenshown to perpetuate the allergic response. Inhibition of NADPH oxidasereduces mast cell degranulation and may be useful in allergic eyedisease (Nishikawa et al, 2007, BBRC, 362 (2): 504-509).

With respect to the role of specific Nox enzymes in inflammatorydisorders, Nox2-containing NADPH oxidase and Akt activation are believedto play a key role in angiotensin II-induced cardiomyocyte hypertrophy(Physiol. Genomics 26: 180-191, 2006).

Accordingly, Nox are believed to be responsible for increased levels ofROS in some cancers and inflammatory disorders, and treatment withappropriate inhibitors may be useful in treating such cancers andinflammatory disorders.

There remains a need for treatment of cancer that does not have theadverse effects generally caused by the non-selectivity of conventionalchemotherapeutic agents. There further remains a need to have additionaltreatments for inflammatory, degenerative and vascular diseases in whicha reactive oxygen species has been implicated. The present inventionprovides such compounds, compositions and methods.

SUMMARY OF THE INVENTION

Compounds, pharmaceutical compositions including the compounds, andmethods of preparation and use thereof are disclosed. The compounds arespecific triphenylmethane compounds, named “imipramine blue” and analogsthereof. The compounds can be formed, for example, by reacting a diarylketone such as Mischler's ketone with imipramine or analogs thereof, inthe presence of a Lewis acid such as phosphorus oxychloride or thionylchloride. Typically, an electrophilic addition occurs at the ortho orpara position to hydroxy or amine groups in the phenol or anilinecompounds, or meta to nitro or carboxy groups, and is followed bydehydration to form the triphenyl methane compounds.

One specific analine is imipramine itself. The analog prepared byreaction with Mischler's ketone is called imipramine blue, and isreferred to in some experiments herein as “TPM2.” The synthesis,characterization and an evaluation of the anti-tumor potential of thesetriarylmethane-containing compounds is also disclosed.

Prodrug forms of the compounds, in which the iminium group on certaintriphenylmethanes are reduced, for example, with sodiumcyanoborohydride, are also disclosed. The prodrugs can readily bereoxidized into the parent compounds, and offer various advantages overthe drugs themselves. For example, the prodrug forms are less coloredand more lipophilic (because the iminium salt is reduced to an amine).The compounds can be more easily taken up by cells than the parentdrugs, and may be less irritating in vivo. In tumors/blood vessels withhigh levels of superoxide/hydrogen peroxide, the prodrugs can be readilyoxidized to the triphenylmethane dye within the cell.

In some embodiments, the reduced compounds (prodrugs) have one or morefree amine groups, which can be reacted with dichloroacetyl chloride tomake one or more trichloroacetyl amide groups. Upon hydrolysis, theprodrugs will hydrolyze in vivo to form dichloroacetic acid salts(“DCA”) and the triphenylmethane compounds, both of which are useful intreating cancer.

While not wishing to be bound by a particular theory, it is believedthat the compounds function by one or more of the following mechanisms:

a) inhibiting all forms of Nox,

b) specifically inhibiting Nox 1-5,

c) specifically inhibiting Nox 2 and/or Nox 4 (the latter of which ismore prevalent in cancer cells than normal cells),

d) inhibiting a Nox enzyme that is more prevalent in cancer cells thannormal cells, hereinafter referred to as tNox,

e) inhibiting ROS,

f) promoting superoxide scavengers, such as scavenger enzyme systemscatalase, superoxide dismutase I (Zn2+/Cu2+SOD) and II (MN-SOD), andglutathione peroxidase, and

inducing G2/M cell cycle arrest.

Evidence that the compounds can inhibit ROS is demonstrated herein inthe working examples, which show that electron spin resonance spectrademonstrate that when the compounds are added to superoxide dismutase,they alter the spectra of the superoxide dismutase, and appear to beconverted to a free radical.

As discussed above, the mechanism for killing the cancer cells mayinvolve inhibition of tNOX, without significantly affecting CNox,thereby effectively inhibit cell proliferation, particularly inmetastasized tumors, or the inhibition of any of the Nox enzymes, suchas Nox4, which is prevalent in cancer cells. That is, in someembodiments, the Nox is one that is selectively expressed in cancercells over normal cells, and in other embodiments, the Nox is one thatis expressed in higher concentrations in cancer cells than in normalcells. In other embodiments, the compounds function by inhibiting theeffects of reactive oxygen species (“ROS”), and/or by inhibiting theproduction of hypoxia inducible factor 2 (HIF2), which drivesangiogenesis due to reactive oxygen.

Treatment with one or more of these compounds can selectively killcancer cells, without killing healthy cells, thus providing a selectiveanti-cancer therapy. Most importantly, these compounds are potentagainst cancer cells that have become metastacized.

Data from WM35 PKB (Stable WM35 cells overexpressing Akt) suggests thatthe compounds can inhibit reactive oxygen in a NOX-independent fashion,for example, by directly scavenging reactive oxygen produced bydefective mitochondria.

By inhibiting ROS and/or HIF2, various inflammatory, degenerative andvascular diseases in which a reactive oxygen species has been implicatedcan be treated.

In another embodiment, the compounds are also effective at treatingparasitic infections, such as malaria, trypanosomiasis, andleishmaniasis, specifically including Chagas and sleeping sickness.

The pharmaceutical compositions include an effective amount of thecompounds described herein, along with a pharmaceutically acceptablecarrier or excipient. When employed in effective amounts, the compoundscan act as a therapeutic agent to prevent and/or treat a wide variety ofcancers, particularly metasticized cancers, and are believed to be bothsafe and effective in this role. Representative cancers that can betreated and/or prevented include melanoma, leukemia, non-small celllung, colon, central nervous system (CNS), renal, ovarian, breast andprostate cancer. Additional pharmaceutical compositions may be usefulfor the treatment of inflammatory, degenerative and vascular diseases inwhich a reactive oxygen species has been implicated.

The foregoing and other aspects of the present invention are explainedin detail in the detailed description and examples set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes a photograph and a chart showing that the inhibition ofang-2 signaling by a soluble tie-2 receptor inhibits bEnd.3 growth invivo. The top panel shows mice with control Fc-treated tumor (left) andtie-2/Fc-treated hemangioma (right). The bottom panel shows thathemangioma volume in mice treated with tie-2/Fc differs significantlyfrom control mice (P<0.05). Three mice were used in each group, and theerror bars represent the standard error of the mean.

FIG. 2 is a graph showing the effectiveness of the imipramine blue(TMP2), at treating Leishmania amazonensis, as measured by the number ofparasites/mL over time (hours). The graphs show results at 0.1, 1.0, 10,and 50 mcM concentrations.

FIG. 3 is a graph showing the effectiveness of ethanol at treatingLeishmania amazonensis, as measured by the number of parasites/mL overtime (hours).

FIG. 4 is a graph showing the effectiveness of imipramine blue attreating Leishmania amazonensis, as measured by the number ofparasites/mL over time (data taken 48 hours after exposure to thecompounds). The graph shows results at 0.05, 0.1, 1, and 5 mcMconcentrations, with ethanol as a control.

FIG. 5 is an ESR spectrum of imipramine blue.

DETAILED DESCRIPTION OF THE INVENTION

Compounds, pharmaceutical compositions including the compounds, andmethods of preparation and use thereof are disclosed.

The following definitions will be useful in understanding the metes andbounds of the invention as described herein.

As used herein, “alkyl” refers to straight chain or branched saturatedhydrocarbon radicals including C₁-C₈, preferably C₁-C₅, such as methyl,ethyl, or isopropyl; “substituted alkyl” refers to alkyl radicalsfurther bearing one or more substituent groups such as hydroxy, alkoxy,aryloxy, mercapto, aryl, heterocyclo, halo, amino, carboxyl, carbamyl,cyano, and the like; “alkenyl” refers to straight chain or branchedhydrocarbon radicals including C₁-C₈, preferably C₁-C₅ and having atleast one carbon-carbon double bond; “substituted alkenyl” refers toalkenyl radicals further bearing one or more substituent groups asdefined above; “cycloalkyl” refers to saturated or unsaturated,non-aromatic, cyclic ring-containing radicals containing three to eightcarbon atoms, preferably three to six carbon atoms; “substitutedcycloalkyl” refers to cycloalkyl radicals further bearing one or moresubstituent groups as defined above; the term “amino” refers to aminegroups bearing zero, one, or two alkyl groups, and includes cyclicamines with ring sizes between three and eight carbons; “aryl” refers toaromatic radicals having six to ten carbon atoms; “substituted aryl”refers to aryl radicals further bearing one or more substituent groupsas defined above; “alkylaryl” refers to alkyl-substituted aryl radicals;“substituted alkylaryl” refers to alkylaryl radicals further bearing oneor more substituent groups as defined above; “arylalkyl” refers toaryl-substituted alkyl radicals; “substituted arylalkyl” refers toarylalkyl radicals further bearing one or more substituent groups asdefined above; “heterocyclyl” refers to saturated or unsaturated cyclicradicals containing one or more heteroatoms (e.g., O, N, S) as part ofthe ring structure and having two to seven carbon atoms in the ring;“substituted heterocyclyl” refers to heterocyclyl radicals furtherbearing one or more substituent groups as defined above.

I. Compounds

The compounds are triarylmethane compounds, or prodrugs or metabolitesof these compounds, and pharmaceutically acceptable salts thereof.Triarylmethane compounds typically include a first carbon-carbon doublebond linked at one end to two aryl or heteroaryl rings, and at the otherend to a cyclohexadiene ring (forming a “first exocyclic double bond”),which optionally includes a second exocyclic double bond, typically inthe form of an imine or ketone group. The two aryl or heteroaryl ringscan be linked via a bridge, which can be, for example, an alkylenebridge, such as a methylene bridge, or a heteroarom, or a direct linkagebetween the rings.

The second exocyclic double bond in the cyclohexadiene can be at aposition “ortho” or “para” to the first exocyclic double bond.

The compounds generally fall within the following formula:

wherein

R is, independently, H, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, heteroaryl, alkylaryl, or arylalkyl, wherein thesubstituent groups are selected from the group consisting of hydroxy,alkoxy, aryloxy, mercapto, aryl, heterocyclo, halo, amino, carboxyl,carbamyl, and cyano;

X is, independently, H, amine, hydroxy, ether, thiol, or thiol ether,and is preferably selected from amine, hydroxy, ether, thiol, and thiolether,

Z=an optional substituent,

Wherein the individual aromatic rings can be substituted at any freeposition with H or a substituent Z, as described herein, and

Substituents defined by Z are selected from the group consisting of C₁₋₆alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl,halo (e.g., F, Cl, Br, or I), —OR′, —NR′R″, —CF₃, —CN, —NO₂, —C₂R′,—SR′, —N₃, —C(═O)NR′R″, —NR′C(═O)R″, —C(═O)R′, —C(═O)OR′, —OC(═O)R′,—OC(═O)NR′R″, —NR′C(═O)OR″, —SO₂R′, —SO₂NR′R″, and —NR′SO₂R″, where R′and R″ are individually hydrogen, C₁₋₆ alkyl, cycloalkyl, heterocyclyl,aryl, or arylalkyl (such as benzyl);

X′ is O, S, NR, or NR₂ ⁺,

any and all salts, prodrugs, racemic mixtures and stereoisomers, andpurified enantiomers of an of the foregoing, and

any and all polymorphs, pseudomorphs, hydrates, and solvates of any ofthe foregoing.

A more specific formula is shown below:

The compounds can occur in varying degrees of enantiomeric excess, andracemic mixtures can be purified using known chiral separationtechniques.

The compounds can be in a free base form or in a salt form (e.g., aspharmaceutically acceptable salts). Examples of suitablepharmaceutically acceptable salts include inorganic acid addition saltssuch as sulfate, phosphate, and nitrate; organic acid addition saltssuch as acetate, galactarate, propionate, succinate, lactate, glycolate,malate, tartrate, citrate, maleate, fumarate, methanesulfonate,p-toluenesulfonate, and ascorbate; salts with an acidic amino acid suchas aspartate and glutamate; alkali metal salts such as sodium andpotassium; alkaline earth metal salts such as magnesium and calcium;ammonium salt; organic basic salts such as trimethylamine,triethylamine, pyridine, picoline, dicyclohexylamine, andN,N′-dibenzylethylenediamine; and salts with a basic amino acid such aslysine and arginine. The salts can be in some cases hydrates or ethanolsolvates. The stoichiometry of the salt will vary with the nature of thecomponents.

The following aryl/heteroaryl rings can be present in the compoundsdescribed herein, as one of the aryl rings in Formula 1.

Representative compounds include the following:

where X is as defined above.

Tautomeric forms of the compounds are also within the scope of theinvention. For example, the compounds shown below can exist in bothtautomeric forms:

Prodrug Forms of the Compounds

In some embodiments, the reduced compounds (prodrugs) have one or morefree amine groups, which can be reacted with dichloroacetyl chloride tomake one or more trichloroacetyl amide groups. Upon hydrolysis, theprodrugs will hydrolyze in vivo to form dichloroacetic acid salts(“DCA”) and the triphenylmethane compounds, both of which are useful intreating cancer. That is, DCA has been tested on in vitro cancer celllines and a rat model, and found to restore mitochondrial function, thusrestoring apoptosis, killing cancer cells in vitro, and shrinking thetumors in the rats. (See Bonnet et al., Cancer Cell 11 (1): 37-51(2007).

For example, the prodrugs can be prepared by reacting quaternaryammonium groups with a reducing agent such as sodium cyanoborohydride toform amines. The amines can be reoxidized in vivo, particularly whentaken up into cancer cells which product reactive oxygen species.

As discussed above, the amine groups can be converted to amide groups,which, upon hydrolysis, yield the amines, which can be oxidized to formthe active compounds. Where the amide group is a trichloroacetyl amidegroup, the hydrolysis also produces the trichloroacetate salt, which canfight cancer via a different route than the active compounds (thusforming an in-situ drug cocktail from a single prodrug. Conditions forforming amides from amines and acids, or acid halides/anhydrides, arewell known to those of skill in the art and need not be repeated here.

II. Methods of Preparing the Compounds

In some embodiments, the compounds can be prepared by reacting a diarylketone with a phenol or aniline compound, in the presence of a Lewisacid or phosphorus oxychloride or thionyl chloride. Using this approach,numerous triphenyl methanes can be made from readily available diarylketones and phenol or aniline starting materials. The reaction proceedsby electrophilic addition of the ketone moiety in the diaryl ketones tothe ortho or para position of the aniline or phenol, followed bydehydration to form the triphenyl methanes.

As discussed below, routine chemistry can be used to prepare the diarylketones.

Methods of Forming Diaryl Ketones

The diaryl ketones used in the coupling step with phenols or anilinescan either be commercially available, such as Michler's ketone, or canbe prepared using known chemistry, or variations of known chemistry.Ideally, the aryl rings are functionalized in a manner that facilitateselectrophilic aromatic substitution reactions (i.e., include electrondonating substituents such as hydroxy, ether, thiol, thiol ether, andamine).

Yun et al., Tetrahedron Letters, Volume 42, Issue 2, Pages 175-177 (8Jan. 2001), discloses using a three-component Stille coupling reactionon solid phase to prepare diaryl ketones bearing a wide variety offunctional groups. The reaction involves using a polymer-boundorganostannane and aryl halides in the presence of carbon monoxide.

Silbestri et al. discloses the synthesis of a series of diaryl ketones,in good yields (40-78%), through the catalyst-free reaction oftrimethylarylstannanes with aroyl chlorides in chlorobenzene as solvent(Silbestri et al., Journal of Organometallic Chemistry, Volume 691,Issue 8, Pages 1520-1524 (1 Apr. 2006)). These reactions are completelyregioselective, making possible the synthesis of diarylketones which arenot usually available under the influence of the directing forces of thesubstituents present in the aromatic ring. Also, the reaction conditionsare mild enough to be applied to acid sensitive molecules.

Duplais et al. (Angewandte Chemie International Edition, Volume 43,Issue 22, Pages 2968-2970 (May 2004)) discloses the efficient synthesisof diaryl ketones by iron-catalyzed arylation of aroyl cyanides.

Enquist et al., Org. Lett., 5 (25), 4875-4878, 2003. 10.1021/o1036091xS1523-7060(03)06091-7 (Nov. 20, 2003), discloses the ultrafast synthesisof diaryl ketones using cobalt carbonyl-mediated synthesis undermicrowave irradiation

The Enquist synthesis combined the advantages of metal activation, insitu carbon monoxide delivery, and microwave heating to efficientlysynthesize benzophenones in 6-10 seconds. These ultrafast carbonylationreactions occur under air by flash heating of aryl iodides in thepresence of dicobalt octacarbonyl. Thus, using suitably functionalizedaryl iodides, one can rapidly prepare desired diaryl ketones.

Any of these reactions can be used to prepare the diaryl ketonesdescribed herein.

Triarylmethane Formation

Examples of the synthesis are shown below in Scheme I (where theasterisks show the respective ortho and para positions on the anilinesand phenols where the electrophilic aromatic addition takes place, andthe values for X and J are those described above with respect toFormulas 1-4):

The compounds can include various aryl and heteroaryl rings, and theseformulas are defined as including the possibility that the rings can befurther functionalized with additional rings (either by direct coupling,as in naphthyl, or by a covalent linkage, as in biphenyl and otherbiaryl rings).

Representative aryl and heteroaryl rings that can be part of the diarylketones, phenols, and anilines are provided below (with theunderstanding that the rings will include appropriate aryl ketone,hydroxy, or amine functionalization):

wherein any of the aryl/heteroaryl rings can be substituted with one ormore substituents as described herein.

Functionalization of the Diaryl Ketones, Phenols and Anilines

The diaryl ketones, phenols, and anilines used to make compoundsdescribed herein are either commercially available, or can be preparedfrom commercially available starting materials. Those that are notcommercially available can be made by a variety of syntheticmethodologies, related to the particular moieties and the particularsubstitution desired. The variation in synthetic methodology will bereadily apparent to those of skill in the art of organic synthesis.

Those skilled in the art will readily understand that incorporation ofother substituents onto the aryl or heteroaryl rings used as a startingmaterial to prepare the triarylmethanes, and other positions in thetriarylmethane framework, can be readily realized. Also, varioussubstituents can be added after the triarylmethanes have been prepared.Such substituents can provide useful properties in and of themselves orserve as a handle for further synthetic elaboration.

Substituents typically can be added to a diaryl ketone before reactionwith the phenol or aniline, or to the aryl ring before preparation ofthe diaryl ketone, as discussed above. One proviso is that suchsubstitution should either survive the triarylmethane synthesis bycoupling the diaryl ketone and the phenol or aniline (i.e., Lewis Acidconditions), or should be added after the triarylmethane synthesis iscomplete.

For example, aryl rings can be halogenated using various knownprocedures, which vary depending on the particular halogen. Examples ofsuitable reagents include bromine/water in concentrated HBr, thionylchloride, pyr-ICI, fluorine and Amberlyst-A. A number of other analogs,bearing substituents in a diazotized position of an aryl ring, can besynthesized from the corresponding aniline compounds, via the diazoniumsalt intermediate. The diazonium salt intermediates can be preparedusing known chemistry, for example, treatment of aromatic amines such asaniline with sodium nitrite in the presence of a mineral acid.

Diazonium salts can be formed from anilines, which in turn can beprepared from nitrobenzenes (and analogous amine-substituted heteroarylrings can be prepared from nitro-substituted heteroaryl rings). Thenitro derivatives can be reduced to the amine compound by reaction witha nitrite salt, typically in the presence of an acid. Other substitutedanalogs can be produced from diazonium salt intermediates, including,but are not limited to, hydroxy, alkoxy, fluoro, chloro, iodo, cyano,and mercapto, using general techniques known to those of skill in theart. For example, hydroxy-triphenyl methane analogues can be prepared byreacting the diazonium salt intermediate with water, protecting theresulting hydroxyl group, forming the cyclopentadienyl anion, andreacting it with a suitable aldehyde or ketone. Likewise, alkoxytriphenyl methane analogues can be made by reacting the diazonium saltwith alcohols. The diazonium salt can also be used to synthesize cyanoor halo compounds, as will be known to those skilled in the art.Mercapto substitutions can be obtained using techniques described inHoffman et al., J. Med. Chem. 36: 953 (1993). The mercaptan so generatedcan, in turn, be converted to an alkylthio substitutent by reaction withsodium hydride and an appropriate alkyl bromide. Subsequent oxidationwould then provide a sulfone. Acylamido analogs of the aforementionedcompounds can be prepared by reacting the corresponding amino compoundswith an appropriate acid anhydride or acid chloride using techniquesknown to those skilled in the art of organic synthesis.

Hydroxy-substituted analogs can be used to prepare correspondingalkanoyloxy-substituted compounds by reaction with the appropriate acid,acid chloride, or acid anhydride. Likewise, the hydroxy compounds areprecursors of both the aryloxy and heteroaryloxy via nucleophilicaromatic substitution at electron deficient aromatic rings. Suchchemistry is well known to those skilled in the art of organicsynthesis. Ether derivatives can also be prepared from the hydroxycompounds by alkylation with alkyl halides and a suitable base or viaMitsunobu chemistry, in which a trialkyl- or triarylphosphine anddiethyl azodicarboxylate are typically used. See Hughes, Org. React.(N.Y.) 42: 335 (1992) and Hughes, Org. Prep. Proced. Int. 28: 127 (1996)for typical Mitsunobu conditions.

Cyano-substituted analogs can be hydrolyzed to afford the correspondingcarboxamido-substituted compounds. Further hydrolysis results information of the corresponding carboxylic acid-substituted analogs.Reduction of the cyano-substituted analogs with lithium aluminum hydrideyields the corresponding aminomethyl analogs. Acyl-substituted analogscan be prepared from corresponding carboxylic acid-substituted analogsby reaction with an appropriate alkyllithium using techniques known tothose skilled in the art of organic synthesis.

Carboxylic acid-substituted analogs can be converted to thecorresponding esters by reaction with an appropriate alcohol and acidcatalyst. Compounds with an ester group can be reduced with sodiumborohydride or lithium aluminum hydride to produce the correspondinghydroxymethyl-substituted analogs. These analogs in turn can beconverted to compounds bearing an ether moiety by reaction with sodiumhydride and an appropriate alkyl halide, using conventional techniques.Alternatively, the hydroxymethyl-substituted analogs can be reacted withtosyl chloride to provide the corresponding tosyloxymethyl analogs,which can be converted to the corresponding alkylaminoacyl analogs bysequential treatment with thionyl chloride and an appropriatealkylamine. Certain of these amides are known to readily undergonucleophilic acyl substitution to produce ketones.

Hydroxy-substituted analogs can be used to prepare N-alkyl- orN-arylcarbamoyloxy-substituted compounds by reaction with N-alkyl- orN-arylisocyanates. Amino-substituted analogs can be used to preparealkoxycarboxamido-substituted compounds and urea derivatives by reactionwith alkyl chloroformate esters and N-alkyl- or N-arylisocyanates,respectively, using techniques known to those skilled in the art oforganic synthesis.

Similarly, benzene rings (and pyridine, pyrimidine, pyrazine, and otherheteroaryl rings) can be substituted using known chemistry, includingthe reactions discussed above. For example, the nitro group onnitrobenzene can be reacted with sodium nitrite to form the diazoniumsalt, and the diazonium salt manipulated as discussed above to form thevarious substituents on a benzene ring.

Synthesis of Imipramine Blue

One novel compound which was tested in several of the examples describedherein has been named “imipramine blue.” Imipramine blue has thefollowing formula:

Imipramine blue can be synthesized by reacting imipramine withMischler's Ketone and an acid like phosphorus oxychloride (POCl₃), asshown below:

Other compounds described herein can be prepared using a similarsynthetic method.

Representative tricyclic moieties other than imipramine that can be usedto prepare the compounds described herein include:

III. Pharmaceutical Compositions

The compounds described herein can be incorporated into pharmaceuticalcompositions and used to prevent a condition or disorder in a subjectsusceptible to such a condition or disorder, and/or to treat a subjectsuffering from the condition or disorder. The pharmaceuticalcompositions described herein include one or more of the triphenylmethane analogues described herein, and/or pharmaceutically acceptablesalts thereof. Optically active compounds can be employed as racemicmixtures, as pure enantiomers, or as compounds of varying enantiomericpurity.

The manner in which the compounds are administered can vary. Thecompositions are preferably administered orally (e.g., in liquid formwithin a solvent such as an aqueous or non-aqueous liquid, or within asolid carrier). Preferred compositions for oral administration includepills, tablets, capsules, caplets, syrups, and solutions, including hardgelatin capsules and time-release capsules. Compositions may beformulated in unit dose form, or in multiple or subunit doses. Preferredcompositions are in liquid or semisolid form. Compositions including aliquid pharmaceutically inert carrier such as water or otherpharmaceutically compatible liquids or semisolids may be used. The useof such liquids and semisolids is well known to those of skill in theart.

The compositions can also be administered via injection, i.e.,intraveneously, intramuscularly, subcutaneously, intraperitoneally,intraarterially, intrathecally; intravitreally, subconjunctivally, andintracerebroventricularly. Intravenous administration is a preferredmethod of injection. Suitable carriers for injection are well known tothose of skill in the art, and include 5% dextrose solutions, saline,and phosphate buffered saline. The compounds can also be administered asan infusion or injection (e.g., as a suspension or as an emulsion in apharmaceutically acceptable liquid or mixture of liquids).

The formulations may also be administered using other means, forexample, rectal administration. Formulations useful for rectaladministration, such as suppositories, are well known to those of skillin the art. The compounds can also be administered by inhalation (e.g.,in the form of an aerosol either nasally or using delivery articles ofthe type set forth in U.S. Pat. No. 4,922,901 to Brooks et al., thedisclosure of which is incorporated herein in its entirety); topically(e.g., in lotion form); or transdermally (e.g., using a transdermalpatch, using technology that is commercially available from Novartis andAlza Corporation). Although it is possible to administer the compoundsin the form of a bulk active chemical, it is preferred to present eachcompound in the form of a pharmaceutical composition or formulation forefficient and effective administration.

The compounds can be incorporated into drug delivery devices such asnanoparticles, microparticles, microcapsules, and the like.Representative microparticles/nanoparticles include those prepared withcyclodextrins, such as pegylated cyclodextrins, liposomes, includingsmall unilamellar vesicles, and liposomes of a size designed to lodge incapillary beds around growing tumors. Suitable drug delivery devices aredescribed, for example, in Heidel J D, et al., Administration innon-human primates of escalating intravenous doses of targetednanoparticles containing ribonucleotide reductase subunit M2 siRNA, ProcNatl Acad Sci USA. 2007 Apr. 3; 104 (14):5715-21; Wongmekiat et al.,Preparation of drug nanoparticles by co-grinding with cyclodextrin:formation mechanism and factors affecting nanoparticle formation, ChemPharm Bull (Tokyo). 2007 March; 55 (3):359-63; Bartlett and Davis,Physicochemical and biological characterization of targeted, nucleicacid-containing nanoparticles, Bioconjug Chem. 2007 March-April; 18(2):456-68; Villalonga et al., Amperometric biosensor for xanthine withsupramolecular architecture, Chem. Commun. (Camb). 2007 Mar. 7;(9):942-4; Defaye et al., Pharmaceutical use of cyclodextrines:perspectives for drug targeting and control of membrane interactions,Ann Pharm Fr. 2007 January; 65 (1):33-49; Wang et al., Synthesis ofOligo(ethylenediamino)-beta-Cyclodextrin Modified Gold Nanoparticle as aDNA Concentrator; Mol Pharm. 2007 March-April; 4 (2):189-98; Xia et al.,Controlled synthesis of Y-junction polyaniline nanorods and nanotubesusing in situ self-assembly of magnetic nanoparticles, J NanosciNanotechnol., 2006 December; 6 (12):3950-4; and Nijhuis et al.,Room-temperature single-electron tunneling in dendrimer-stabilized goldnanoparticles anchored at a molecular printboard, Small. 2006 December;2 (12):1422-6.

Exemplary methods for administering such compounds will be apparent tothe skilled artisan. The usefulness of these formulations may depend onthe particular composition used and the particular subject receiving thetreatment. These formulations may contain a liquid carrier that may beoily, aqueous, emulsified or contain certain solvents suitable to themode of administration.

The compositions can be administered intermittently or at a gradual,continuous, constant or controlled rate to a warm-blooded animal (e.g.,a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, or monkey),but advantageously are administered to a human being. In addition, thetime of day and the number of times per day that the pharmaceuticalformulation is administered can vary.

Preferably, the compositions are administered such that activeingredients interact with regions where cancer cells are located. Thecompounds described herein are very potent at treating these cancers.

In certain circumstances, the compounds described herein can be employedas part of a pharmaceutical composition with other compounds intended toprevent or treat a particular cancer, i.e., combination therapy. Inaddition to effective amounts of the compounds described herein, thepharmaceutical compositions can also include various other components asadditives or adjuncts.

Complexation with Proteins

The compounds described herein can be complexed with peptides andproteins, including albumin, transferrin, VEGF, bFGF, and the like.These complexes are easy to make and tend to have lower toxicity thanthe un-complexed compounds.

When administered, it is believed that the triphenylmethanes are highlyprotein bound. One can create novel delivery forms by taking advantageof this binding, in that aqueous solutions of triphenylmethane can beincubated with specific proteins that can target leaky vessels (iealbumin) or proteins that target the tumor or its vasculature (VEGF,transferrin, collagen type 7, insulin like growth factor, PDGF, etc).Once the triphenylmethane has formed a complex with the protein, it canbe infused, for example, intraveneously, intramuscularly, and/orsubcutaneously, and the protein complex binds to the receptor, isinternalized, and the triphenylmethane is released in the target cell.

Combination Therapy

The combination therapy may be administered as (a) a singlepharmaceutical composition which comprises a triphenyl methane analogueas described herein, at least one additional pharmaceutical agentdescribed herein, and a pharmaceutically acceptable excipient, diluent,or carrier; or (b) two separate pharmaceutical compositions comprising(i) a first composition comprising a triphenyl methane analogue asdescribed herein and a pharmaceutically acceptable excipient, diluent,or carrier, and (ii) a second composition comprising at least oneadditional pharmaceutical agent described herein and a pharmaceuticallyacceptable excipient, diluent, or carrier. The pharmaceuticalcompositions can be administered simultaneously or sequentially and inany order.

In use in treating or preventing cancer, the triphenyl methane analoguesdescribed herein can be administered together with at least one otherchemotherapeutic agent as part of a unitary pharmaceutical composition.Alternatively, the triphenyl methane analogues can be administered apartfrom the other anticancer chemotherapeutic agent. In this embodiment,the triphenyl methane analogues and the at least one other anticancerchemotherapeutic agent are administered substantially simultaneously,i.e. the compounds are administered at the same time or one after theother, so long as the compounds reach therapeutic levels for a period oftime in the blood.

Combination therapy involves administering a triphenyl methane analogue,as described herein, or a pharmaceutically acceptable salt or prodrug ofa compound described herein, in combination with at least oneanti-cancer chemotherapeutic agent, ideally one which functions by adifferent mechanism (i.e., VEGF inhibitors, alkylating agents, and thelike).

Examples of known anticancer agents which can be used for combinationtherapy include, but are not limited to alkylating agents, such asbusulfan, cis-platin, mitomycin C, and carboplatin; antimitotic agents,such as colchicine, vinblastine, paclitaxel, and docetaxel; topo Iinhibitors, such as camptothecin and topotecan; topo II inhibitors, suchas doxorubicin and etoposide; RNA/DNA antimetabolites, such as5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites,such as 5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine;and antibodies, such as Herceptin® and Rituxan®. Other known anti-canceragents, which can be used for combination therapy, include arsenictrioxide, gamcitabine, melphalan, chlorambucil, cyclophosamide,ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin,bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoicacid, tamoxifen and alanosine. Other classes of anti-cancer compoundsthat can be used in combination with the triphenyl methane analogues aredescribed below.

The triphenyl methane analogues can be combined withalpha-1-adrenoceptor antagonists, such as doxazosin, terazosin, andtamsulosin, which can inhibit the growth of prostate cancer cell viainduction of apoptosis (Kyprianou, N., et al., Cancer Res 60:4550 4555,(2000)).

Sigma-2 receptors are expressed in high densities in a variety of tumorcell types (Vilner, B. J., et al., Cancer Res. 55: 408 413 (1995)) andsigma-2 receptor agonists, such as CB-64D, CB-184 and haloperidol,activate a novel apoptotic pathway and potentiate antineoplastic drugsin breast tumor cell lines. (Kyprianou, N., et al., Cancer Res. 62:313322 (2002)). Accordingly, the triphenyl methane analogues can becombined with at least one known sigma-2 receptor agonists, or apharmaceutically acceptable salt of said agent.

The triphenyl methane analogues can be combined with lovastatin, aHMG-CoA reductase inhibitor, and butyrate, an inducer of apoptosis inthe Lewis lung carcinoma model in mice, can potentiate antitumor effects(Giermasz, A., et al., Int. J. Cancer 97:746 750 (2002)). Examples ofknown HMG-CoA reductase inhibitors, which can be used for combinationtherapy include, but are not limited to, lovastatin, simvastatin,pravastatin, fluvastatin, atorvastatin and cerivastatin, andpharmaceutically acceptable salts thereof.

Certain HIV protease inhibitors, such as indinavir or saquinavir, havepotent anti-angiogenic activities and promote regression of Kaposisarcoma (Sgadari, C., et al., Nat. Med. 8:225 232 (2002)). Accordingly(in addition to forming triphenyl methane analogues of these compounds),the triphenyl methane analogues can be combined with HIV proteaseinhibitors, or a pharmaceutically acceptable salt of said agent.Representative HIV protease inhibitors include, but are not limited to,amprenavir, abacavir, CGP-73547, CGP-61755, DMP-450, indinavir,nelfinavir, tipranavir, ritonavir, saquinavir, ABT-378, AG 1776, andBMS-232,632.

Synthetic retinoids, such as fenretinide (N-(4-hydroxyphenyl)retinamide,4HPR), can have good activity in combination with other chemotherapeuticagents, such as cisplatin, etoposide or paclitaxel in small-cell lungcancer cell lines (Kalemkerian, G. P., et al., Cancer Chemother.Pharmacol. 43:145 150 (1999)). 4HPR also was reported to have goodactivity in combination with gamma-radiation on bladder cancer celllines (Zou, C., et al., Int. J. Oncol. 13:1037 1041 (1998)).Representative retinoids and synthetic retinoids include, but are notlimited to, bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, .alpha.-difluoromethylornithine, ILX23-7553, fenretinide, andN-4-carboxyphenyl retinamide.

Proteasome inhibitors, such as lactacystin, exert anti-tumor activity invivo and in tumor cells in vitro, including those resistant toconventional chemotherapeutic agents. By inhibiting NF-kappaBtranscriptional activity, proteasome inhibitors may also preventangiogenesis and metastasis in vivo and further increase the sensitivityof cancer cells to apoptosis (Almond, J. B., et al., Leukemia 16:433 443(2002)). Representative proteasome inhibitors include, but are notlimited to, lactacystin, MG-132, and PS-341.

Tyrosine kinase inhibitors, such as STI571 (Imatinib mesilate,Gleevec®), have potent synergetic effects in combination with otheranti-leukemic agents, such as etoposide (Liu, W. M., et al. Br. J.Cancer 86:1472 1478 (2002)). Representative tyrosine kinase inhibitorsinclude, but are not limited to, Gleevec®, ZD1839 (Iressa®), SH268,genistein, CEP2563, SU6668, SU11248, and EMD121974.

Prenyl-protein transferase inhibitors, such as farnesyl proteintransferase inhibitor R115777, possess antitumor activity against humanbreast cancer (Kelland, L. R., et. al., Clin. Cancer Res. 7:3544 3550(2001)). Synergy of the protein farnesyltransferase inhibitor SCH66336and cisplatin in human cancer cell lines also has been reported (Adjei,A. A., et al., Clin. Cancer. Res. 7:1438 1445 (2001)). Prenyl-proteintransferase inhibitors, including farnesyl protein transferaseinhibitor, inhibitors of geranylgeranyl-protein transferase type I(GGPTase-I) and geranylgeranyl-protein transferase type-II, or apharmaceutically acceptable salt of said agent, can be used incombination with the triphenyl methane analogues described herein.Examples of known prenylprotein transferase inhibitors include, but arenot limited to, R115777, SCH66336, L-778,123, BAL9611 and TAN-1813.

Cyclin-dependent kinase (CDK) inhibitors, such as flavopiridol, havepotent, often synergetic, effects in combination with other anticanceragents, such as CPT-11, a DNA topoisomerase I inhibitor in human coloncancer cells (Motwani, M., et al., Clin. Cancer Res. 7:4209 4219,(2001)). Representative cyclin-dependent kinase inhibitors include, butare not limited to, flavopiridol, UCN-01, roscovitine and olomoucine.

Certain COX-2 inhibitors are known to block angiogenesis, suppress solidtumor metastases, and slow the growth of implanted gastrointestinalcancer cells (Blanke, C. D., Oncology (Hunting) 16 (No. 4 Suppl. 3):1721 (2002)). Representative COX-2 inhibitors include, but are not limitedto, celecoxib, valecoxib, and rofecoxib.

IκB-α phosphorylation inhibitors, such as BAY-11-7082 (an irreversibleinhibitor of IκB-α phosphorylation) are also known to induce apoptosis,or to enhance the effectiveness of other agents at inducing apoptosis.These inhibitors can also be used in combination with the compoundsdescribed herein.

Any of the above-mentioned compounds can be used in combination therapywith the triphenyl methane analogues. Additionally, many of thesecompounds can be converted to triphenyl methane analogues by reaction ofketone, aldehyde, hydroxyl, thiol, and/or amine functional groups on thecompounds using the chemistry described herein. The triphenyl methaneanalogues of these compounds are within the scope of this invention.

Further, the triphenyl methane analogues can be targeted to a tumor siteby conjugation with therapeutically useful antibodies, such asHerceptin® or Rituxan®, growth factors, such as DGF, NGF; cytokines,such as IL-2, IL-4, or any molecule that binds to the cell surface. Theantibodies and other molecules will deliver a compound described hereinto its targets and make it an effective anticancer agent. Thebioconjugates can also enhance the anticancer effect of therapeuticallyuseful antibodies, such as Herceptin® or Rituxan®.

The compounds can also be used in conjunction with surgical tumorremoval, by administering the compounds before and/or after surgery, andin conjunction with radiation therapy, by administering the compoundsbefore, during, and/or after radiation therapy.

The appropriate dose of the compound is that amount effective to preventoccurrence of the symptoms of the disorder or to treat some symptoms ofthe disorder from which the patient suffers. By “effective amount”,“therapeutic amount” or “effective dose” is meant that amount sufficientto elicit the desired pharmacological or therapeutic effects, thusresulting in effective prevention or treatment of the disorder.

When treating cancers, an effective amount of the triphenyl methaneanalogue is an amount sufficient to suppress the growth of the tumor(s),and, ideally, is a sufficient amount to shrink the tumor, and, moreideally, to destroy the tumor. Cancer can be prevented, eitherinitially, or from re-occurring, by administering the compoundsdescribed herein in a prophylactic manner. Preferably, the effectiveamount is sufficient to obtain the desired result, but insufficient tocause appreciable side effects.

The effective dose can vary, depending upon factors such as thecondition of the patient, the severity of the cancer, and the manner inwhich the pharmaceutical composition is administered. The effective doseof compounds will of course differ from patient to patient, but ingeneral includes amounts starting where desired therapeutic effectsoccur but below the amount where significant side effects are observed.

The compounds, when employed in effective amounts in accordance with themethod described herein, are selective to certain cancer cells, but donot significantly affect normal cells.

For human patients, the effective dose of typical compounds generallyrequires administering the compound in an amount of at least about 1,often at least about 10, and frequently at least about 25 μg/24hr/patient. The effective dose generally does not exceed about 500,often does not exceed about 400, and frequently does not exceed about300 μg/24 hr/patient. In addition, administration of the effective doseis such that the concentration of the compound within the plasma of thepatient normally does not exceed 500 ng/mL and frequently does notexceed 100 ng/mL.

IV. Methods of Using the Compounds and/or Pharmaceutical Compositions

The compounds described herein, and pharmaceutical compositionsincluding the compounds, can be used to treat cancers. The cancersinclude those in which one of the Nox enzymes is present in elevatedconcentrations (i.e., Nox 1, Nox 4, and the like), or those in whichcancer growth is mediated by ROS.

Representative disorders that can be treated include neoplasms, such ashemangiomas, and malignant tumors, for example, those which arise in thesetting of autocrine loops involving vascular endothelial growth factor(VEGF) and its major mitogenic receptor vascular endothelial growthfactor receptor 2. Representative malignant tumors include malignantendothelial tumors such as melanoma.

Representative malignant tumors include malignant endothelial tumorssuch as melanoma. Additional cancers that can be treated include, butnot limited to human sarcomas and carcinomas, e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., plasma cell leukemia, acute lymphocyticleukemia, acute myelocytic leukemia (myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia); chronic leukemia(chronic myelocytic (granulocytic) leukemia and chronic lymphocyticleukemia); and polycythemia vera, lymphoma (Hodgkin's disease andnon-Hodgkin's disease), including NF-KB mutant and Velcade Resistantlymphoma cells, multiple myeloma, PI3 kinase deficient myeloma,Waldenstrom's macroglobulinemia, and heavy chain disease, and malignantforms of these cancers. Additionally, the compounds can be used inassays involving lymphoblastoid and EBV positive cells.

In one embodiment, the cancer is melanoma, rectal carcinoma, coloncarcinoma, breast carcinoma, ovarian carcinoma, small cell lungcarcinoma, colon carcinoma, chronic lymphocytic carcinoma, hairy cellleukemia, esophogeal carcinoma, prostate carcinoma, breast cancer,myeloma, or lymphoma. It is believed that these cancers have circulatinglevels of tNOX (which may include Nox4 or other Nox enzymes) present inthe sera of patients suffering from the cancer (see, for example, U.S.Pat. No. 5,605,810, which is hereby incorporated by reference in itsentirety).

In some embodiments, the patient already has cancer and is undergoingtreatment for the cancer, and may or may not have tumor metastasis(i.e., secondary cancer).

In other embodiments, the compounds are active at inhibitinghypoxia-inducible factor HIF2a expression, and this activity aids in thetreatment of tumors resistant to standard chemoradiotherapy.Hypoxia-inducible factor HIF2alpha (HIFalphas) regulates the expressionof a variety of genes encoding proteins related to angiogenesis and toanaerobic metabolism of cells exposed to hypoxic stress (Koukourakis etal., Int J Radiat Oncol Biol Phys. 2002 Aug. 1; 53 (5):1192-202.) HIF2aoverexpression is significantly associated with high microvessel density(p=0.02, respectively) and with VEGF expression (p=0.005), andVEGF/KDR-activated tumor vasculature is more frequent inHIF2a-overexpressing tumors (p=0.02). High HIF2a levels have beenassociated with incomplete response to chemoradiation (p=0.02,respectively), and overexpression of HIF2a is related to locallyaggressive behavior, intensification of angiogenesis, and resistance tocarboplatin chemoradiotherapy.

Imipramine blue was evaluated in a HIF2a expression model, and was shownto inhibit around 90% of the HIF2a expression. In this expression model,WM35 PKB cells are exposed to 5 micromolar of test compounds for 24hours. At the end of this period, cells are harvested for RNA, which isthen reverse transcribed into cDNA, and levels of HIF2a message arequantified using quantitative RT-PCR and corrected for a housekeepingRNA message. As demonstrated using this assay, these compounds can actas direct NADPH oxidase inhibitors, as well as superoxide scavengersthat absorb superoxide produced by defective mitochondria or othercellular processes.

The cancer may be manifested in the form of a tumor, such as a tumor ofepithelial tissue, lymphoid tissue, connective tissue, bone, or centralnervous system.

The compounds can also be used as adjunct therapy in combination withexisting therapies in the management of the aforementioned types ofcancers. In such situations, it is preferably to administer the activeingredients to in a manner that optimizes effects upon cancer cells,including drug resistant cancer cells, while minimizing effects uponnormal cell types. While this is primarily accomplished by virtue of thebehavior of the compounds themselves, this can also be accomplished bytargeted drug delivery and/or by adjusting the dosage such that adesired effect is obtained without meeting the threshold dosage requiredto achieve significant side effects.

Specific Disorders Mediated by Nox₂ and Nox₄ Receptors

Nox₂ and Nox₄ are NADPH oxidases. Nox₂ exists as part of a multiproteincomplex, known as cytochrome b558, which includes phox p47phox, p22phoxand p21rac, as well as p67phox. Rac may be either rac1 or 2. Nox₄ maynot require complexing with these proteins.

Nox₂ is highly expressed in neutrophils, macrophages and lymphocytes,and may mediate reactive oxygen driven NFkB in inflammatory andneoplastic processes involving neutrophils, macrophages, andlymphocytes, ie leukemias and lymphomas.

Nox4 is a widely distributed NADPH oxidase, which is highly expressed inmany malignancies, ie melanoma, pancreatic cancer, etc, and generatesreactive oxygen that drives NFkB, which then activates antiapoptoticgenes such as bc12 and mcl-1, resulting in resistance to radiation andchemotherapy.

It is proposed that blockade of superoxide will result in decreasedexpression of NFkB, and will sensitize malignancies to radiation andchemotherapy.

Disorders that are characterized by excess reactive oxygen can beclassified into disorders which can be treated and prevented, andpatients whose immune systems become more active followingadministration of the compounds described herein (i.e., immunepotentiation). The following disorders can be treated using thecompounds described herein:

a) Neoplastic-hemangiomas, melanoma, lung cancer, breast cancer, coloncancer, pancreatic cancer, prostate cancer, ovarian cancer, brain tumorsincluding glioblastoma multiforme, sarcomas, head and neck cancers,hepatocellular carcinoma, nasopharyngeal carcinoma, cervical cancer andprecancer, hematologic malignancies including multiple myeloma,myelodysplatic syndrome, acute and chronic leukemias, Hodgkins disease,non hodgkins lymphoma, including diffuse large B cell lymphoma (DLBCL)

b) Inflammatory disorders-psoriasis, atopic dermatitis (eczema), asthma,arthritis (including rheumatoid, psoriatic), inflammatory bowel disease(including Crohns and ulcerative colitis), lupus, multiple sclerosis,Sjogrens disease, gastritis and pernicious anemia, sprue, sarcoidosis,vitiligo, alopecia areata, scleroderma, fibrotic disorders, cirrhosis,coronary artery disease, atherosclerosis, myositis, myocarditis

c) Degenerative disease-macular degeneration, Alzheimers disease,parkinsons disease, lewy body dementia, prion mediated disorders,emphysema, cataracts, photoaging of skin, pterygium, osteoporosis,pattern hair loss, hypertension, stroke

D) Infectious disease-Bacterial, including Staph, i.e. MRSA, viral(i.e., herpes, HIV, HBV, HCV, HPV, and influenza), and fungal (i.e.,Candida)

Treatment of Osteoporosis

The compounds described herein can also be used to treat osteoporosis.The cytokine RANKL (receptor activator of NF-κB ligand) causesosteoporosis by activating osteoclasts. The compounds inhibit RANKLactivity by potentiating apoptosis, suppresses osteoclastogenesis, andinhibits invasion through modulation of nuclear factor-kappaB activationpathway (see, for example, Mol Cancer Res. 2006 September; 4(9):621-33).

Treatment of Inflammatory Disorders

The compounds described herein are useful for treating or preventinginflammatory disorders. Reactive oxygen drives NFkB in inflammatorydisorders such as rheumatoid arthritis, asthma, psoriasis, excema,lupus, scleroderma, certain heart diseases such atherosclerosis andcoronary artery disease, and the like. Because the compounds areeffective at inhibiting production of reactive oxygen species, they areactive against inflammatory disorders.

The compounds also inhibit certain inflammatory signals, and canalleviate inflammatory disorders such as inflammatory arthritis byinhibiting these signals.

Rheumatoid arthritis (RA) is considered the most common systemicautoimmune disease, but other disorders, such as hypothyroidism,systemic lupus erythematosus (SLE), and the like can also be treatedusing the compounds described herein. A number of conditions areassociated with chronic inflammation and elevated levels of TNF-α andIL-6, including rheumatoid arthritis, heart disease, and cancer.Numerous gastrointestinal disorders are caused by inflammation,including, but not limited to, Crohn's disease, irritable bowelsyndrome, and inflammatory bowel syndrome, and these disorders can alsobe treated and/or prevented using the compounds described herein.

There is a suggested link between rheumatoid arthritis and chronicinflammation due to the re-activation of Epstein-Barr virus (EBV), whichlatently infects a proportion of memory B cells in >90% of the world'spopulation. Among the EBV-encoded proteins implicated in viralpathogenesis, considerable attention has focused upon latent membraneprotein 1 (LMP1). Of the nine EBV genes expressed as proteins inEBV-transformed cells, LMP1 is the best characterized, and is the onlyEBV-encoded gene product capable of transforming cells in vitro and invivo, resulting in the potential for lymphoproliferative changes andmalignancy. In addition to its established role in the pathogenesis of Bcell lymphoma and other malignancies, EBV infection may be linked toexacerbation of various human autoimmune diseases, including RA and SLE.

The mouse collagen-induced arthritis (CIA) model (Myers, et al., LifeScience 61: 1861-1878 (1997)) has many pathologic and immunologicparallels to rheumatoid arthritis, and provides a stable, predictablemodel for evaluating the therapeutic potential of compounds for treatingchronic inflammatory conditions. This model can be used, for example, toevaluate the ability of the compounds described herein to treat and/orprevent these disorders.

Treatment of mouse B cell lines with compounds described herein in vitrocan be shown to recapitulate the cytokine profile seen in primary mouseB cells with a concomitant dose-dependent decrease in CD40 andLMP1-mediated NFkB and AP-1 activation. Those compounds which decreaseCD40 and LMP1-mediated NFkB and AP-1 activation in a dose-dependentmanner will be expected to have anti-inflammatory properties,potentially in both the cognitive phase of the immune response, as wellas the effector phase, by inhibiting cytokines that lead to chronicinflammation and additional pathology.

Treatment of Neurodegenerative Disorders and/or ProvidingNeuroprotection

Reactive oxygen species also induce inflammation and neurodegeneration.Inhibition of these species can also result in neuroprotection,including protection from further damage following an ischemic braininjury such as a stroke, or that caused from blunt trauma, and treatmentor prevention of neurodegenerative disorders such as retinaldegenerations, Alzheimer's disease, senile dementia, pre-seniledementia, Parkinsons disease, Fragile X syndrome, tuberous sclerosis,Huntington's Chorea, multiple sclerosis, and the like.

Reactive oxygen species also drive seizures, and the compounds mayameliorate seizures as well.

Treatment of Vascular Disorders

Vascular diseases such as erectile dysfunction and migraines in whichROS have been implicated may also respond to NADPH oxidase inhibitors.The compounds described herein can be used to treat these vasculardiseases.

Atherosclerosis is one vascular disorder known to be treated with NADPHoxidase inhibitors (see, for example, U.S. Pat. No. 5,763,496).Accordingly, the compounds can be used to prevent atherosclerosis and/orinhibit the development of an atherosclerotic plaque.

Treatment of Parasitic Infections

Certain of the compounds described herein can be used to treat orprevent parasitic certain infections. Among those parasitic infectionsthat can be treated include malaria, trypanosomiasis, and leishmaniasis.

Malaria is caused by protozoan parasites of the genus Plasmodium. Thereare four species of Plasmodium, Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale, and Plasmodium malaria. P. falciparum is the mostwidespread and dangerous, and left untreated, it can lead to fatalcerebral malaria.

Leishmaniasis is a disease caused by protozoan parasites belonging tothe genus Leishmania. The disease is transmitted by the bite of certainspecies of sand flies, including flies in the genus Lutzomyia in the NewWorld and Phlebotomus in the Old World.

There are four main forms of leishmaniasis. Visceral leishmaniasis isthe most serious form and potentially fatal if untreated. Cutaneousleishmaniasis is the most common form, and causes a sore at the bitesite. The sore can heal in a few months to a year, leaving an unpleasantlooking scar, but can also progress to any of the other three forms.Diffuse cutaneous leishmaniasis is a form that produces widespread skinlesions which resemble leprosy and is particularly difficult to treat.Mucocutaneous leishmaniasis begins with skin ulcers which spread,causing tissue damage to the nose and mouth.

Trypanosomiasis is a disorder caused by a parasite called a trypanosome,and is commonly known as sleeping sickness. The parasite is transmittedto humans through the bite of a tsetse fly. There are two forms ofAfrican sleeping sickness, caused by two different parasites.Trypanosoma brucei gambiense causes a chronic infection lasting years,and is largely found in western and central Africa. Trypanosoma bruceirhodesiense causes acute illness lasting several weeks, and is largelyfound in eastern and southern Africa. If untreated, trypanosomiasiscauses tremendous suffering, and ultimately ends in death.

Certain of the compounds described herein are effective at treating oneor more of these disorders.

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, allparts and percentages are by weight, unless otherwise noted. Reactionyields are reported in mole percentages.

EXAMPLES

The following examples are provided to illustrate the present inventionand should not be construed as limiting the scope thereof. In theseexamples, all parts and percentages are by weight, unless otherwisenoted. Reaction yields are reported in mole percentage.

Example 1 Spectrophotometric Assay of NADH Oxidase

NADH oxidase activity can be determined as the disappearance of NADHmeasured at 340 nm in a reaction mixture containing 25 mM Tris-Mesbuffer (pH 7.2), 1 mM KCN, and 150 μM NADH at 37° C. Activity can bemeasured, for example, using a Hitachi U3210 spectrophotometer withstiffing and continuous recording over two intervals of 5 mM each. Amillimolar extinction coefficient of 6.22 can be used to determinespecific activity.

Example 2 Measuring Cell Growth

A mouse mammary tumor subpopulation line 4T1 arising from a BALB/cf C3Hmouse can be grown in DME-10, Dulbecco's modified Eagle's mediumsupplemented with 5% fetal calf serum, 5% newborn calf serum, 1 mM mixednon-essential amino acids, 2 mM L-glutamine, penicillin (100 units/ml),and streptomycin (100 μg/ml) (Miller et al., 1987, Brit. J. Can.56:561-569 and Miller et al., 1990, Invasion Metastasis 10:101-112).

Example 3 Pharmacologic Blockade of Angiopoietin-2 is EfficaciousAgainst Model Hemangiomas in Mice

Hemangioma of infancy is the most common neoplasm of childhood. Whilehemangiomas are classic examples of angiogenesis, the angiogenic factorsresponsible for hemangiomas are not fully understood. Malignantendothelial tumors arise in the setting of autocrine loops involvingvascular endothelial growth factor (VEGF) and its major mitogenicreceptor vascular endothelial growth factor receptor 2.

Hemangiomas of infancy differ from malignant endothelial tumors in thatthey usually regress, or can be induced to regress by pharmacologicmeans, suggesting that angiogenesis in hemangiomas differs fundamentallyfrom that of malignant endothelial tumors.

The data in this example demonstrate constitutive activation of theendothelial tie-2 receptor in human hemangioma of infancy and, using amurine model of hemangioma, bEnd.3 cells. bEnd.3 hemangiomas produceboth angiopoietin-2 (ang-2) and its receptor, tie-2, in vivo. Inhibitionof tie-2 signaling with a soluble tie-2 receptor decreases bEnd.3hemangioma growth in vivo. The efficacy of tie-2 blockade suggests thateither tie-2 activation or ang-2 may be required for in vivo growth.

To address this issue, tie-2-deficient bEnd.3 hemangioma cells wereused. Surprisingly, these cells were fully proficient in in vivo growth.Previous studies from our laboratory and others have implicated reactiveoxygen-generating nox enzymes in the angiogenic switch, so the effect ofnox inhibitors was evaluated on ang-2 production in vitro and on bEnd.3tumor growth in vivo. Ang-2 production was inhibited pharmacologicallyusing novel inhibitors of nox enzymes, and this treatment nearlyabolished bEnd.3 hemangioma growth in vivo. Signal-transduction blockadetargeting ang-2 production may therefore be useful in the treatment ofhuman hemangiomas in vivo.

The following abbreviations are used throughout this example:

Ang, angiopoietin;

DPI, diphenyliodonium;

VEGF, vascular endothelial growth factor

Introduction

Hemangiomas are the most common tumor of infancy and childhood andaccount for a disproportionate number of visits to pediatricians anddermatologists (Chiller et al., 2003). Histologically, hemangiomasconsist of clusters of endothelial cells surrounding vascular lumens ofvarying diameter. The natural history of hemangiomas begins with aproliferative phase, characterized by rapid growth of the tumor andendothelial division, followed by an involuting stage, which is markedby endothelial apoptosis and decreasing tumor size, and finally endswith an involuted stage, during which the original tumor is replaced bya connective tissue scar (Takahashi et al., 1994). While these tumorsusually resolve spontaneously, large tumors can compromise the functionof vital organs by compression, and may even lead to high-output cardiacfailure (Drolet et al, 1999).

Studies have established the clonal nature of hemangiomas and suggestedthat growth factors may play a role in the pathogenesis of hemangiomas(Boye et al, 2001; Yu et al., 2001). Our laboratory has previously shownthat autocrine production of vascular endothelial growth factor (VEGF)by endothelial cells results in malignant transformation to angiosarcoma(Arbiser et al., 2000; McLaughlin et al., 2000). Therefore, wepostulated that another factor, which acts as an endothelialchemoattractant and survival factor, is responsible for autocrine growthin hemangiomas. Here, we demonstrate that the tie-2 receptor isconstitutively phosphorylated in human hemangioma, implicating eitherconstitutive activation of tie-2 or deregulated production ofangiopoietin-2 (ang-2) as a causative factor in hemangiomagenesis. Toelucidate the functional role of these agents in the pathogenesis ofhemangioma, we used a murine model of hemangioma, bEnd.3 cells (Bautchet al., 1987; Williams et al., 1989), and found that these cells expressboth tie-2 and its ligand, ang-2. We also demonstrate that functionalblockade of tie-2 using a soluble receptor inhibits the growth of bEnd.3hemangiomas in vivo. Surprisingly, tie-2-deficient endothelial cellswere also capable of initiating hemangiomas in vivo, implicatingaberrant ang-2 production as a potential cause of hemangiomas.Nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (Nox)genes have previously been linked to the angiogenic switch, and havebeen known to regulate ang-2 (Arbiser et al., 2002; Krikun et al.,2002). As we were unable to inhibit ang-2 stably using small interferingRNA, we discovered novel inhibitors of ang-2 production through blockadeof Nox genes. These inhibitors nearly abolish bEnd.3 hemangioma growthin vivo. Thus, our data suggest that neutralization of ang-2 through Noxinhibition may be an effective therapy for hemangiomas of infancy.

Results

Tie-2 and ang-2 are highly expressed in bEnd.3 hemangiomas in vivo

In order to determine whether bEnd.3 hemangiomas exhibit potentialautocrine loops involving tie-2 and angs, we performed in situhybridization of lesions in mice. We found that bEnd.3 hemangiomasexhibit expression of both ang-2 and tie-2. Vascular endothelial growthfactor receptor 1 and 2 were expressed at high levels, consistent withactive endothelial remodeling, and small quantities of VEGF mRNA wereobserved. Use of control sense probes for VEGF did not revealhybridization. No significant hybridization for ang-1 was observed (datanot shown).

Inhibition of ang-2 Using a Soluble Receptor Inhibits bEnd.3 Growth InVivo

To determine whether inhibition of ang-2 expression by blockade of itsreceptor, tie-2, was needed for hemangioma formation in vivo, bEnd.3cells were infected with adenoviruses encoding either soluble tie-2/Fcor adenoviruses encoding Fc fragment alone, 24 hours before injection.This treatment resulted in an approximately 66% decrease in tumorvolume, compared to control adenovirus treatment (FIG. 1). No toxicitywas observed as a consequence of infection in any of the three animalsthat were used in each group. Attempts to generate stably infectedbEnd.3 cells with lentiviral small interfering RNA for ang-2 wereunsuccessful, possibly owing to a requirement for ang-2/tie-2 signalingfor longer term growth.

Tie-2 is not Required for In Vivo Growth of Hemangiomas

Given that functional blockade of the tie-2 receptor significantlyreduced hemangioma formation in vivo, we wanted to determine whetherthis was the result of impaired ang-2 signaling or tie-2 inhibition.Polyoma-expressing endothelial cells derived from tie-2-deficient micewere injected into nude mice, and compared to controls, no significantdifference in tumor volume or histology was noted. Efforts to generatebEnd.3 clones expressing small interfering RNA to ang-2 wereunsuccessful, perhaps owing to severe growth disadvantages.

Example 4 Analysis of Triarylmethanes in Treating Parasitemia

A series of in vivo experiments were performed to evaluate the abilityof the triarylmethanes, in particular, imipramine blue, to treatparasites, such as trypanosomes. The bloodstream forms of many, but notall, trypanosomes (a type of parasite) solely depend on trypanosomealternative oxidase (TAO), for respiration. Accordingly, those of thecompounds described herein which can inhibit the TAO can effectivelykill the parasite.

Gentian violet has been used as a trypanocidal agent for Chagas disease.The data presented herein show that it is also possible to inhibit Tbrucei oxidases using Gentian violet, analogues thereof, and othercompounds described herein.

In one study, the parasite was T. brucei. Mice were injected withparasites (5,000 parasites/mouse), and injected (s.c) with imipramineblue (1 mg/mice/day) starting from the day the mice were injected withparasites. The parasitemia level in blood was counted on each followingday.

The results showed that both the control and the imipramine-treated micedied at day 4 because of the parasite load. However, the number of theparasite/ml of blood were slightly less in the imipramine-treated mice.

The experiment was repeated with IV administration of imipramine blue(100 μg of imipramine blue). Because the parasite (T. brucei) grows inthe blood and tissue fluid and does not enter into any cells, it wasbelieved that IV administration could effectively target the parasites.This treatment resulted in a 70% decrease in parasitemia in the mousemodel. However, a dosage of 1 mg IV killed the mice. Accordingly, it isimportant to find a therapeutic window (where treatment of parasitemiacan be effected, without unwanted toxic effects). This therapeuticwindow is in the range of between about 150 and 250 μg.

Imipramine blue also showed favorable cell toxicity to trypanosometoxicity at 500 nm. These and other compounds were tested on live cells.

The experiment was conducted with one million cells of the bloodstreamform of T. brucei. After over night treatment with different compounds(at concentrations of 50 and 100 μmolar), cells were counted and plottedwith controls. All cells died in gentian violet and brilliant green.

The results are as follows:

The IC₅₀ value for Imipramine Blue (IP) was 50<100 nM.

Example 5 Treatment of Malaria Using Triarylmethanes

Imipramine blue, a triarylmethane analogue of imipramine, is achemosensitizer, and useful in the treatment of malaria Imipramine blue,as well as other triarylmethanes described herein, also has NADPHoxidase inhibitor activity, and some of these compounds have shownactivity against trypanosomes and leishmania. While not wishing to bebound to a particular theory, it is believed that these compounds havedual activity against malaria through chemosensitization and inhibitionof Plasmodium NADPH oxidase.

The compounds were tested in a malaria model using both chloroquinesensitive and multidrug resistant P. falciparum. The malaria model wasthe same as published in JAC Advance Access published online on Jul. 30,2008, Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn315, thecontents of which are hereby incorporated by reference, and the data issummarized in the table below.

Briefly, the assay involves exposing both chloroquine sensitive andmultidrug resistant P. falciparum to the compounds. After one hour ofexposure to the highest concentration of the compound, followed byremoval of the compound, the growth of all stages of P. falciparum wasobserved. The reduction in the growth stages was then compared withuntreated control parasites. Any stage-specific effects were noted atany of the concentrations. The presence of strong inhibition (definedherein as 10% growth) of all parasite stages was evaluated when theparasites were exposed to various concentrations of various compounds.

IC₅₀ (nM) vs P. falciparum D6 Dd2 Compound (Chloroquine Sensitive)(Multidrug Resistant) Chloroquine Control 7.9 139.6 Imipramine Blue (IB)~2,500 825.8

The data show that imipramine blue has some activity in this assay.

Example 6 Treatment of Leishmaniasis

In addition to the other parasitic diseases discussed in other examples,the compounds were also tested in a leishmaniasis model.

The experiments were started using water as a solvent for thetriarylmethanes (TPM, standing for triphenylmethane). However, water wasnot an effective solvent, so ethanol was substituted. Ethanol is toxicto leishmania, so a control group was tested with a higher ethanolconcentration than that present on the TPM groups. The results of theexperiment are presented in FIGS. 4-8.

FIG. 2 has five with 50, 10, 1 and 0.1 mcM (micromolar) of TPM2(imipramine blue) in ethanol, and a positive control.

Imipramine blue was found to have activity, but ethanol also was active.Accordingly, a separate experiment was conducted to determine how toxicthe ethanol is without added compounds. Concentrations of 5, 1 and 0.1%of ethanol were used. FIG. 3 shows the effect of ethanol alone.

At the lower concentration (0.1% ethanol), no toxicity was observed atthe first 48 hours. Thus, the results obtained in the first experimentwith the last two concentrations of imipramine blue in ethanol (1 and0.1 mcM), where the concentration of ethanol were 0.1 and 0.01%, can beaccepted (the dark blue and pink lines on the first four graphics).Thus, TPM 2 has shown activity in these concentrations.

To confirm these data, another experiment was performed with TPM 2 with1; 0.5; 0.1 and 0.05 mcM, where the ethanol was present in a non-toxicconcentration. The number of parasites was counted only after 48 hours.Again, an ethanol control at 0.1% was used.

With this new experiment, it was found that TPM2 has significantactivity at 1 mcM, where the growth inhibition was 72%. The IC₅₀ wasaround 0.3 mcM for TPM1 and 0.6 mcM for TPM 2.

The IC₅₀ was calculated by the method described by Hills et al. (18) andHuber and Koella (19). Briefly, Hills proposed finding twoconcentrations, X₁ and X₂, such that the parasite density, Y1, atconcentration X₁ (and all lower concentrations) was more than half ofthe density found in the control, Y₀, and that the parasite density, Y₂,at concentration X₂ (and all higher concentrations) was less than halfof Y₀. The IC₅₀ was then found by linear extrapolation between X₁ andX₂:

log(IC₅₀)=log(X ₁)+[(Y ₁ −Y ₀/2)/(Y ₁ −Y ₂)][log(X ₂)−log(X ₁)].

Preliminary data showed activity against in vitro promastigotes (theflagellate stage of a trypanosomatid protozoan).

The results of all of these experiments are summarized in the followingtable.

Experiment TPM MW IC₅₀ 1 and 2 TPM 2 588.87 0.6 μM

Example 7 Use of Imipramine Blue to Induce Apoptosis and Cell CycleArrest in Myeloma Cells—Mediated in a p53 Dependent Pathway

Background:

Tricyclic antidepressant drugs imipramine, clomipramine and citalopraminduce neurogenesis (1) and are also known to induce apoptosis in cancercells (2). However, the molecular mechanism underlying the therapeuticeffects of these drugs in various intracellular signaling pathways andpossibly a clinical utility of one these compounds in cancer treatmenthas not been well explored. Imipramine blue has been recently shown toinhibit CDK inhibitor p21 expression and subsequent release of neuronalprogenitor cells from the blockade of proliferation (3). Impramine bluetreatment induces extensive DNA damage in cultured C6 rat glioma cells(4) and induces cell growth inhibition (5).

Aim:

The aim of this investigation was to determine the capacity ofimipramine blue in cell growth inhibition and its potency to induceapoptosis in myeloma cells as well as in various hematological cancercell types.

Materials and Methods:

Imipramine blue was obtained from the department of dermatology, EmoryUniversity. MTT assays for myeloma cells (MM.1S, MM.1R, RPMI8226, andU266) were used to evaluate the cell viability. AnnexinV staining andcell cycle analysis was done by flow cytometry to assess the level ofapoptosis and analyze cell cycle arrest. Western blotting was performedusing antibodies to analyze the impact of imipramine blue on varioustargets. Bone marrow from myeloma patients were obtained with consentfrom the Winship Cancer Institute.

Results:

When myeloma cells were treated up to 72 hrs, the cell viability assaysshowed that treatment of imipramine blue up to 10 μM results in morethan 80% growth inhibition effectively between 6 uM to 10 μM. Apoptosisassay by annexin V staining shows that the cells were induced to undergoapoptosis with similar concentrations. Procaspases (caspase 8, 9, 3 andPARP) were extensively cleaved from a low concentration as low as 0.5 μMto 10 μM imipramine blue in about 48 hrs. DNA damage was observed basedon the elevated levels of phos-p53 and GADD45 in MM.1S cells. The cellcycle profile indicated that imipramine blue induces both cell cyclearrests as well as apoptosis in MM.1S cells. Combining imipramine blueeither with bortezomib or perifosine suggested that there is additivebenefit in combination. Imipramine blue overcomes the growth advantageby cytokines IL-6 and IGF1 in MM.1S cells. Imipramine blue induced cellgrowth inhibition in both leukemia and in lymphoma cell lines. Primarycells obtained from myeloma patients showed a very significant cellkilling of CD38 positive population by imipramine blue, either alone asa single agent or in combination with bortezomib. The phos-AKT was notsignificantly reduced while both phos-p53 and GADD45 were upregulaed asa result of imipramine blue treatment in MM.1S.

Conclusion:

Imipramine blue has a potential to induce apoptosis and cell cyclearrest in both myeloma as well as in primary myeloma cells, and also inother hematological cancer cell types. The cellular response seems to bemainly through DNA damage, apoptosis, and cell cycle arrest. Imipramineblue is believed to have clinical utility in the treatment ofhematological cancer.

REFERENCES

-   1. The antidepressants imipramine, clomipramine, and citalopram    induce apoptosis in human AML (HL-60) cells via caspase-3 action.    Xia et. al. J. Biochem Mol. Toxicology. 1999; 13; 338-347-   2. Effects of imipramine on ion channels and proliferation of IGR1    melonoma cells. Journal of Membr Biol. 2002; 188:137-149-   3. p21 ^(Cip)1 restricts neuronal proliferation in the subgranular    zone of the dentate gyrus of the hippocampus. Pechnick, et al. 2008;    PNAS; 105, 1358-1363-   4. Assessment of DNA damage in C6 glioma cells after antidepressant    treatment using alkaline comet assay. Slamon et. al. Arch. Toxicol.    2001; 75; 243-250-   5. Chronic Treatment With Imipramine Inhibits Cell growth and    Enhances Serotonin 2C Receptor mRNA Expression in NG 108-15 Cells.    Sukma, et. al. Pharmacol Sci. 2003:92, 433-436

Example 8 ESR Spectrum of Imipramine Blue and Superoxide Dismutase

Li used ESR to confirm the production of NADPH-dependent .O2—by isolatedendosomes (Li et al., Molecular and Cellular Biology, January 2006, p.140-154, 26 (1):140-154 (2006)). ESR assays were conducted at roomtemperature using a Bruker model EMX ESR spectrometer (Bruker).Vesicular fractions from each sample were mixed with the spin trap, 50mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO), in a total volume of 500 μlof PBS, pH 7.4. This solution contained iminodiacetic acid-chelatingresin (10 ml/liter; Sigma-Aldrich). The reaction was initiated by addingNADPH to 100 μM and was immediately placed into the ESR spectrometer.DMPO-hydroxyl radical adduct formation was assayed for 10 min Instrumentsettings were as follows: receiver gain, 1×10⁶; modulation frequency,100 kHz; microwave power, 40.14 mW; modulation amplitude, 1.0 G; andsweep rate, 1 G/s.

In the instant application, the ESR spectrum of imipramine blue and ofsuperoxide dismutase were taken using conditions substantially asdescribed in Li et al. The ESR spectra (FIG. 5) show that imipramineblue appears to form a radical by reacting with superoxide, thusinhibiting the ability of superoxide dismutase to generate ROS.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A compound of the following formula:

wherein R is, independently, H, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, or arylalkyl, wherein thesubstituent groups are selected from the group consisting of hydroxy,alkoxy, aryloxy, mercapto, aryl, heterocyclo, halo, amino, carboxyl,carbamyl, and cyano; X is, independently, H, amine, hydroxy, ether,thiol, or thiol ether, and is preferably selected from amine, hydroxy,ether, thiol, and thiol ether, X′ is O, S, NR, or NR₂ ⁺, Z=an optionalsubstituent, Wherein the individual aromatic rings can be substituted atany free position with H or a substituent Z, as described herein, andSubstituents defined by Z are selected from the group consisting of C₁₋₆alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl,halo (e.g., F, Cl, Br, or I), —OR′, —NR′R″, —CF₃, —CN, —NO₂, —C₂R′,—SR′, —N₃, —C(═O)NR′R″, —NR′C(═O)R″, —C(═O)R′, —C(═O)OR′, —OC(═O)R′,—OC(═O)NR′R″, —NR′C(═O)OR″, —SO₂R′, —SO₂NR′R″, and —NR′SO₂R″, where R′and R″ are individually hydrogen, C₁₋₆ alkyl, cycloalkyl, heterocyclyl,aryl, or arylalkyl; and pharmaceutically acceptable salts and prodrugsthereof, wherein the compounds can be present as racemic mixtures,stereoisomers, or purified enantiomers, and wherein the compounds can bepresent in the form of hydrates and solvates.
 2. the compound of claim1, having the formula:

wherein X and X′ are as defined in claim
 1. 3. The compound of claim 1,having the formula:


4. A method for treating a cancer in a mammal having cancer cells thatexpress tNOX, comprising administering to the mammal a therapeuticallyeffective amount of a composition comprising a compound of claim 1 in anamount sufficient to inhibit a therapeutically significant amount oftNOX expression, such that the growth of the tumor is at least partiallyinhibited.
 5. The method of claim 4, wherein the compound is a compoundof claim
 2. 6. The method of claim 4, wherein the compound is imipramineblue or a pharmaceutically-acceptable salt or prodrug thereof.
 7. Themethod of claim 4 wherein the mammal is a human.
 8. The method of claim4 wherein the cancer is selected from a group consisting of hemangioma,melanoma, rectal carcinoma, colon carcinoma, breast carcinoma, ovariancarcinoma, small cell lung carcinoma, colon carcinoma, chroniclymphocytic carcinoma, hairy cell leukemia, osophogeal carcinoma,prostate carcinoma, breast cancer, myeloma, and lymphoma.
 9. The methodof claim 4, wherein the cancer is a metastasized cancer.
 10. The methodof claim 4 wherein the human is immunosuppressed by reason of havingundergone anti-cancer therapy prior to administration of thecomposition.
 11. The method of claim 4, wherein the cancer is manifestedin the form of a solid tumor having cells that express tNOX.
 12. Themethod of claim 11, wherein the tumor is a tumor of epithelial tissue,lymphoid tissue, connective tissue, bone, or the central nervous system.13. The method of claim 4, wherein the compounds are administeredparenterally, orally, or directly into the tumor.
 14. The method ofclaim 4, wherein the compounds are administered via an implanted device.15. The method of claim 4, wherein the administration is provided usinga sustained release formulation.
 16. A method for treating parasiticinfections in a mammal, comprising administering to the mammal atherapeutically effective amount of a compound of claim 1 in an amountsufficient to treat the disorder.
 17. The method of claim 16, whereinthe amount of the triphenyl methane therapeutic agent is sufficient toinhibit a therapeutically significant amount of NADPH oxidase.
 18. Themethod of claim 17 wherein the mammal is a human.
 19. The method ofclaim 17 wherein the parasitic infection is selected from the groupconsisting of malaria, trypanosomiasis, and leishmaniasis.
 20. Themethod of claim 17, wherein the administration is provided using asustained release formulation.